F NMR Reveals the Dynamics of Substrate Binding and Lid Closure for Iodotyrosine Deiodinase as a Complement to Steady-State Kinetics and Crystallography.

Active site lids are common features of enzymes and typically undergo conformational changes upon substrate binding to promote catalysis. Iodotyrosine deiodinase is no exception and contains a lid segment in all of its homologues from human to bacteria. The solution-state dynamics of the lid have now been characterized using F NMR spectroscopy with a CF-labeled enzyme and CFO-labeled ligands. From two-dimensional F-F NMR exchange spectroscopy, interconversion rates between the free and bound states of a CFO-substituted tyrosine (45 ± 10 s) and the protein label (40 ± 3 s) are very similar and suggest a correlation between ligand binding and conformational reorganization of the lid. Both occur at rates that are ∼100-fold faster than turnover, and therefore these steps do not limit catalysis. A simple CFO-labeled phenol also binds to the active site and induces a conformational change in the lid segment that was not previously detectable by crystallography. Exchange rates of the ligand (130 ± 20 s) and protein (98 ± 8 s) in this example are faster than those above but remain self-consistent to affirm a correlation between ordering of the lid and binding of the ligand. Both ligands also protect the protein from limited proteolysis, as expected from their ability to stabilize a compact lid structure. However, the minimal turnover of simple phenol substrates indicates that such stabilization may be necessary but is not sufficient for efficient catalysis.